11 3.1. INTRODUCTION MATERIALS AND METHODS Materials Methods for solubilty measurements Methods for Liquid Anti-Solvent (LAS) crystallization Characterization methods for particles in suspension Characterization methods for dried powder RESULTS AND DISCUSSION ON SOLUBILITY STUDY Measurement and correlation of solubility of CRS 74 in water-ethanol mixtures Experimental determination Correlation of solubility data by a UNIQUAC model Theoritical yield of solid obtained by LAS crystallization RESULTS AND DISCUSSION ON CRYSTALLIZATION STUDY Characterization of particles in suspension Influence of the type of mixer on mixing Influence of the type of mixer on particle size Influence of flow rate in the T- mixer on particle size Influence of addition time and steady state Characterization of solid particles obtained in experimental design Comparative study of original CRS 74 and LAS recrystallized drug with T-mixer Particle size and morphology XRD analysis DSC analysis Dissolution studies Determination of surface properties CONCLUSIONS CHAPTER INTRODUCTION Materials and methods Materials Methods Production of particles by LAS crystallization in presence of additives Determination of solubility in presence of additive Characterization methods Measurements of particle size during the crystallization process

12 Powder physicochemical properties RESULTS AND DISCUSSION CRS 74 solubility in presence of additives Effect of additives on yields of production of CRS 74 crystals Effect of presence of the additive in the aqueous phase on physical and surface properties of recrystallized powders Effect of the presence of the additive in the organic phase on physical and surface properties of recrystallized powders Effect of the presence of the additive in both, aqueous and organic phases Drug concentration in the organic phase Reorganized organic phase Improving process production Dissolution behaviour of recrystallized powders in presence of additives CONCLUSION CHAPTER APPENDIXES SYMBOLS AND ABBREVIATIONS LIST OF FIGURES AND TABLES REFERENCES

13 Abstract This study concerns a new antiretroviral drug named CRS 74. This molecule has a limited bioavailability because of its low aqueous solubility, poor water wettability and low dissolution rate. In an attempt to improve these properties, CRS 74 was recrystallized by using a Liquid Anti-Solvent (LAS) crystallization process. The chosen solvent is the ethanol and the anti-solvent the water. So solid-liquid equilibria in binary mixtures ethanol/water were measured at 30 C. The obtained solubility data were represented using UNIQUACbased model. The experimental and calculated solubilities permitted to estimate the optimal ethanol/water mass ratios (25/75 % w/w) in order to maximize the theoretical yield of solid. A double-jet with premixing (T-mixer) has been used to mix the two solutions. Particles of recrystallized CRS 74 seemed more agglomerated and the dissolution profile was not modified compared to the original drug. Furthermore, the study of crystals obtained at the exit of the mixer showed that the growth and agglomeration rates of crystals are high.in an attempt to improve its dissolution properties, CRS 74 has been recrystallized using different additives to optimize process and formulation parameters. Conclusively, produced microcrystals exhibited significantly faster dissolution rates than the original CRS 74 crystals. The improved dissolution is attributable to the modification of the particle size of drug crystals and enhancement of wetting properties due to specific interactions between the drug and the additives. Keywords: antiretroviral drug, solubility, bioavailability, dissolution rate, Liquid Anti Solvent crystallization, additive

19 Introduction INTRODUCTION An increasing number of newly developed drugs are poorly soluble in aqueous media. Poorly water soluble drugs are specially challenging, as they cannot achieve dissolution and therefore they have a very difficult pass through the dissolving fluid to contact the absorbing mucosa and to be absorbed. If the dissolution process of the drug molecule is slow, due to the physicochemical properties of the drug molecules or formulation factors, then dissolution may be the rate-limiting step in absorption and will influence drug bioavailability. This is the case of a new compound, (2S, 3S, 5S)-2, -5 bis- [N-[N-[[N- methyl- N-[(2-isopropyl- 4- tiazolyl) methyl] amino] carbonyl] vanilyl] amino- 1,6- diphenyl- 3- hydroxyhexane, named CRS 74. This promising candidate against HIV infections has high biological activity as disclosed in PCT documents WO 2005/111006; US 2010/ (BOCKELMANN, M.A. et al, 2005; BOCKELMANN, M.A. et al, 2010) but its bioavailability is limited because of its low aqueous solubility and dissolution rate. Such properties pose difficulties not only in the design of pharmaceutical formulations but may result in biovariability. Moreover, because of their low solubility, such drugs require high doses to be administered in order to obtain their pharmacological effect, increasing the side effect incidence. Drug dissolution is a prerequisite to drug absorption and clinical response for almost all drugs given orally. Solid forms that have been investigated for drug dissolution enhancement include salts, polymorphs and amorphous, among others. High energy polymorphs and amorphous formulations can achieve improved solubility but the system is at serious risk of crystallizing the thermodynamically stable form, even in the solid state (YU, 2011; RODRIGUEZ-SPONG et al., 2004). Such transformations can compromise the performance of the formulation. To save time and resources in product development, relatively simple approaches should be tried first like crystallization. The Liquid Anti-Solvent (LAS) crystallization process is an attractive method. It requires mild conditions (ambient temperature and atmospheric pressure) with no requirement for expensive equipment. In LAS process, crystallization of solute is achieved by decreasing the solubility of solid in the system. This is 19

20 Introduction done by addition of a non-solvent component for solute called anti-solvent and miscible with the solvent. This thesis presents efforts to develop and assemble tools required for improving the dissolution rate of CRS 74 by LAS crystallization process. It is divided into five chapters. Chapter 1 is devoted to review literature themes. Many strategies to increase the dissolution rate of poorly soluble drugs and the key determinants of drug bioavailability are presented. It also focuses on topics of interest for our work, like properties of drugs influencing dissolution behavior. Emphasis was given to the LAS crystallization process. The first elements of the understanding of the drug properties are given in Chapter 2. The original (as-received) molecule was characterized in terms of its physical properties (particle shape and size), crystal structure (crystalline, amorphous) and surface properties (wettability), solubility and dissolution properties in aqueous media. The methodology utilized and the results obtained are summarized in this Chapter. This characterization study was crucial to evaluate possible changes on drug properties after recrystallization by LAS process. The solvent selection is one of the essential parameters to envisage any crystallization process. Therefore, the knowledge of the solubility of a target component in different solvents is required. The solubility of CRS 74 in ethanol and ethanol-water binary mixtures was measured in the temperature range of 5-30 o C and this study is presented in Chapter 3. Moreover, Chapter 3 outlines the experimental apparatus and procedures used for LAS crystallization studies. Two high jet velocity devices were tested to provide adequate mixing to incorporate the anti-solvent into the bulk solution. Recrystallized solids were compared to the original drug crystals in terms of particle size, solid state, thermal and dissolution properties. Finally, LAS crystallization of CRS 74 in presence of additives is the subject of the Chapter 4. The study describes the use of different additives, which were introduced in the drug solution or in the anti-solvent or in both phases, to achieve optimum crystal properties of CRS 74. The effect of additives on the crystals particle size, dissolution kinetics and drug wettability could be investigated and are discussed in this Chapter. Chapter 5 summarizes the main results of this research and suggests future work. 20

27 Literature Review 1.1. THE BIOAVAILABILITY OF DRUGS Absorption and bioavailability The term bioavailability is usually defined as the rate and extent of absorption of a drug from its dosage form into the systemic circulation (BLANCHARD and SAWCHUK, 1979). By definition, when a medication is administered intravenously, its bioavailability is 100%. However, when a medication is administered via other routes (such as orally), its bioavailability (oral bioavailability) is usually less than 100%, caused by degradation or metabolism of the drug prior to absorption, incomplete absorption and first-pass metabolism not seen with intravenous administration (OSCIER et al., 2007). The therapeutic effect of drugs depends on the drug concentration at the site of action. The absorption of the drug into the systemic circulation is a prerequisite to reach the site of action for all drugs, except those drugs that are applied at the site of action, or those that are intravenously injected. When a drug is taken orally administration (gastrointestinal route) it passes through the mouth, esophagus, stomach, duodenum, jejunum (small intestine), colon (large intestine) (see Figure 1.1) and finally leaves the body if not absorbed. Indeed, it must withstand the effect of several physiological fluctuations like a large variation in ph along the gastrointestinal tract (Table 1.1), the presence of bile salts, food, enzymes, bacteria and the motility of the gut. Esophagus Duodenum Stomach Small intestine Colon Appendix Colon Rectum Figure 1.1. Anatomy of digestive tract (Modified from: MARTINEZ et al., 2002). 27

28 CHAPTER 1 Table 1.1. Transit time and ph conditions along the GI tract (MARTINEZ et al., 2002). GI segment Transit time ph Stomach 2 h 2.0 ± 1.9 Duodenum 10 min 6.6 ± 0.5 Jejunum 2 h 7.4 ± 0.4 Ileum 1 h 7.5 ± 0.4 Colon h 7.0 ± 0.7 Absorption of drugs after oral administration of pharmaceutical dosage forms (drug powder, tablet, capsule ) may occur at the various body sites between the mouth and rectum. After oral administration, the pharmaceutical product reaches quickly the stomach passing through esophagus. However, the small intestine has the largest surface for drug absorption in GI tract (PODCZECK et al. 2007). After disintegration of the pharmaceutical dosage form in the gastrointestinal tract, the first requirement for absorption is that the drug dissolves. In fact, only drug that is dissolved has the ability to permeate the intestinal membrane. The oral bioavailability of a particular drug is thus determined by the magnitude of the solubility and/or permeability limitations that exist for it within the GI tract, which is an aqueous environment. These two aspects, illustrated in Figure 1.2, form the basis of the Biopharmaceutical Classification System (BCS), which is incorporated in the guidelines of the Food and Drug Administration (FDA) established by AMIDON et al. (1995) and often cited in the literature. According to the BCS, four different types of drug absorption regimes are distinguished. They are explained in Table 1.2. According to Biopharmaceutical Classification System (BCS), Class I drugs dissolve rapidly in an aqueous environment and are rapidly transported over the absorbing membrane. For Class II drugs, the dissolution rate in vivo is usually the rate limiting step in drug absorption. Drugs of this group are poorly water soluble. Class III drugs dissolve readily, but cannot penetrate biomembranes of GI tract. 28

30 CHAPTER 1 It is clear that, depending on the classification of the drug, different strategies can be applied to increase or accelerate the absorption of a drug taken orally, either increasing the amount of dissolved drug or increasing the permeability of the dissolved drug through the absorbing membrane. To sum up, Class I drugs do not need a formulation strategy to increase the absorption. The rate of absorption of Class II drugs can be enhanced by accelerating the dissolution. This has proven to be effective in many studies (YELLELA, 2010; PATEL et al., 2011; VIÇOSA et al., 2012). The strategy for Class III drugs is to increase the permeability of the absorbing membrane. Numerous studies deal with increasing membrane permeability in the gastrointestinal tract (NEELAM et al., 2012; SHAIKH et al., 2012). For a Class IV drug, both dissolution as well as permeability must be increased. Over 90% of the marketed drugs qualify under Class II and Class IV (GRIFFIN. 2012). The oral bioavailability of these drug compounds is limited due to slow drug dissolution in the gastrointestinal tract. Therefore, it is desirable to improve the solubility of these drug compounds by using various pharmaceutical technologies that will be discussed later in this Chapter Poorly-water soluble drug molecules Poorly-water soluble drugs describe a heterogeneous group of drug compounds that exhibit poor solubility in water but are typically soluble in various organic solvents. The degree of water solubility for drug compounds can be defined as slightly soluble (1-10mg/mL), very slightly soluble (0.1-1 mg/ml), and practically insoluble (<0.1mg/mL) (MENG, 2011) The expression poorly-water soluble drug generally refers to a drug whose aqueous solubility falls into the range of slightly soluble and below. Examples of commonly marketed drugs that are poorly soluble or insoluble in water include analgesics, cardiovasculars, hormones, antivirals, immune suppressants and antibiotics (BERGESE, 2003). A drug with improved water solubility can be administrated in a lower concentrated dose, with a reduction 30

Absorption of Drugs Absorption is the transfer of a drug from its site of administration to the bloodstream. The rate and efficiency of absorption depend on the route of administration. For IV delivery,

General Certificate of Education Advanced Level Examination June 2012 French Unit 4 Speaking Test Candidate s Material To be conducted by the teacher examiner between 7 March and 15 May 2012 (FRE4T) To

Design and Development of Conventional and Modified Release Oral Drug Delivery Systems Three Day Intensive Course for Managers, Scientists and Technicians with the Emphasis on the Principles of Oral Drug

CHEMISTRY STANDARDS BASED RUBRIC ATOMIC STRUCTURE AND BONDING Essential Standard: STUDENTS WILL UNDERSTAND THAT THE PROPERTIES OF MATTER AND THEIR INTERACTIONS ARE A CONSEQUENCE OF THE STRUCTURE OF MATTER,

Chapter 13 - Solutions 13-1 Types of Mixtures I. Solutions A. Soluble 1. Capable of being dissolved B. Solution 1. A homogeneous mixture of two or more substances in a single phase C. Solvent 1. The dissolving

Overview The standards for chemistry establish scientific inquiry skills and core content for all chemistry courses in DoDEA schools. In chemistry, students acquire a fundamental knowledge of the substances

THE PROPERTIES AND STRUCTURE OF MATTER COURSE CONTENT 1. Define matter and state of matter 2. Properties of solids, liquids and gases 3. Changes in matter Physical and chemical changes Phase changes of

Texas University Interscholastic League Contest Event: Science (Chemistry) The contest challenges students to read widely in chemistry, to understand the significance of experiments rather than to recall

Chemistry UNIT I: Introduction to Chemistry The student will be able to describe what chemistry is and its scope. a. Define chemistry. b. Explain that chemistry overlaps many other areas of science. The

Chapter 14 Solutions 1 14.1 General properties of solutions solution a system in which one or more substances are homogeneously mixed or dissolved in another substance two components in a solution: solute

#171 Guidance for Industry Waivers of In Vivo Demonstration of Bioequivalence of Animal Drugs in Soluble Powder Oral Dosage Form Products and Type A Medicated Articles (This version of the guidance replaces

Percentage Ladder French Unit 1: Qu est-ce que tu aimes regarder? Year 8 and Percentage I can Prove it! 80% I can understand authentic spoken texts and material from a range of voices. I can paraphrase

Terms and Equations of Reverse Osmosis There is a set of terms and equations used to define the parameters governing transport across a membrane. This paper presents the concepts of Reverse Osmosis and

AP FRENCH LANGUAGE 2008 SCORING GUIDELINES Part A (Essay): Question 31 9 Demonstrates STRONG CONTROL Excellence Ease of expression marked by a good sense of idiomatic French. Clarity of organization. Accuracy

s 1 Measurement and Problem Solving 2-3 Weeks Measurements are subject to errors which need to be accounted for. 1. How do you accurately measure, using significant figures, in the metric system? Understand

FOR TEACHERS ONLY The University of the State of New York REGENTS HIGH SCHOOL EXAMINATION F COMPREHENSIVE EXAMINATION IN FRENCH Friday, June 16, 2006 1:15 to 4:15 p.m., only SCORING KEY Updated information

Q 10 Method of Shelf-life estimation Q 10 approach is an old concept that could be useful for estimating the shelf-life at room temperature of products recommended for cold storage. Calculations are based

Chemistry Objectives Matter, and Measurement 1. know the definition of chemistry and be knowledgeable 3-14 about specific disciplines of chemistry. 2. understand the nature of the scientific method and

Research Article ISSN: 2349 4492 Asian Journal of Research in Biological and Pharmaceutical Sciences Journal home page: www.ajrbps.com IMPROVEMENT OF SOLUBILITY OF OMEPRAZOLE MAGNESIUM BY SOLID DISPERSION

Chemistry B11 Chapter 6 Solutions and Colloids Solutions: solutions have some properties: 1. The distribution of particles in a solution is uniform. Every part of the solution has exactly the same composition

I. MIXTURES: SOLUTIONS 1) mixture = a blend of two or more kinds of matter, each of which retains its own identity and properties a) homogeneous mixture = a mixture that is uniform in composition throughout

POLYMORPHISM OF DRUGS CAN WE EXPLOIT PHYSICAL FORM IN THE DEVELOPMENT OF LOW SOLUBILITY MOLECULES? Sponsored by www.crystalpharmatech.com Presented by ELIZABETH B. VADAS, PhD InSciTech Inc. Montreal, Canada

Knowledge Subject Knowledge Audit - French Meta-linguistic challenges full some none When using common verbs in the Present tense, recognise patterns & make analogies. Contrast Present in French with the

pharmahub Collaboration for Pharmaceutical Engineering and Science Linas Mockus Ann Christine Catlin Purdue University What is pharmahub? pharmahub: a cyber infrastructure developed at Purdue University

Guidance for Industry Food-Effect Bioavailability and Fed Bioequivalence Studies U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER)

A11 HEALTH CARE DIRECTIVES ACT Advances in medical research and treatments have, in many cases, enabled health care professionals to extend lives. Most of these advancements are welcomed, but some people

Emulsion Aggregation Toner Technology In my article in this publication in a previous issue, I described the first commercial process for manufacturing chemically prepared toner. In this article I will

LESSON ASSIGNMENT LESSON 4 Emulsions and Suspensions. LESSON ASSIGNMENT Paragraphs 4-1 through 4-14. LESSON OBJECTIVES After completing this lesson, you should be able to: 4-1. Given a group of definitions,

EMULSIONS, SUSPENSIONS AND OTHER DISPERSE SYSTEMS Nahed HEGAZY, PhD A disperse system consists essentially of one component, the disperse phase, dispersed as particles or droplets throughout another component,

Microencapsulation & Delivery systems A new activity at SEPPIC January 2010 Introduction: Definitions Microencapsulation Isolation of an active ingredient from the external environment (protection of the

PROGRAM SEMESTER SUBJECT BOOK ID MSc/PGDiploma in Clinical Research and Regulatory Affairs MCR - BASICS OF PHARMACY, DRUG DISCOVERY AND DEVELOPMENT B709 SESSION FALL 0 No Question/Answer key Marks Total

Nursing 113 Pharmacology Principles 1. The study of how drugs enter the body, reach the site of action, and are removed from the body is called a. pharmacotherapeutics b. pharmacology c. pharmacodynamics

NUNAVUT HOUSING CORPORATION - BOARD MEMBER RECRUITMENT The is seeking Northern Residents interested in being on our Board of Directors We are seeking individuals with vision, passion, and leadership skills

(EPR) Analysis of in the EU and development of guiding principles for their functioning In association with: ACR+ SITA LUNCH DEBATE 25 September 2014 Content 1. Objectives and 2. General overview of in

Nanoparticle Technologies 1 2 NANOPARTICLE SYNTHESIS FROM BATCH TO CONTINUOUS The importance and unique advantages of nano- and microparticles made from different types of inorganic and organic/polymeric

THE DEVELOPMENT OF OFFICE SPACE AND ERGONOMICS STANDARDS AT THE CITY OF TORONTO: AN EXAMPLE OF SUCCESSFUL INCLUSION OF ERGONOMICS AT THE DESIGN STAGE BYERS JANE, HARDY CHRISTINE, MCILWAIN LINDA, RAYBOULD

4. Thermodynamics of Polymer Blends Polymeric materials find growing applications in various fields of everyday life because they offer a wide range of application relevant properties. Blending of polymers